When whole blood is fixed at clinical sites using the TokuKit, can samples be thawed, aliquoted, re-frozen at −80 °C, and later re-analyzed without impacting data quality or results?
We set out to answer this directly, because for translational and clinical teams, the ability to maximize the value of limited clinical samples is critical.
We evaluated refreeze performance using whole blood from a healthy donor under two conditions:
Stimulations were used to induce expression of functional markers that would not be detectable at baseline. Stimulated samples were prepared using a defined blend of PMA/PTI-activated whole blood spiked with PHA-stimulated PBMCs and Daudi cells, followed by fixation with the TokuKit to “lock in” functional states. This enabled us to directly evaluate whether functional markers (activation, exhaustion, proliferation, cytotoxicity, etc.) induced prior to freezing are retained following refreeze
Samples were tested across three conditions:
After initial fixation and storage at −80 °C, refreeze cycles were simulated by thawing, washing, counting, aliquoting, and refreezing samples in 10% Dimethyl sulfoxide (DMSO) /Cell Staining Buffer (CSB) at −80 °C. All samples were ultimately thawed and processed in the same run using a 43-marker mass cytometry panel to ensure direct comparability.
Across up to two freeze–thaw cycles, immune cell population frequencies were highly stable:
Functional marker subset frequencies also remained consistent after refreezing:
These results demonstrate that both lineage-level and functional biology are preserved.
This refreeze-validated workflow is fully supported through Teiko’s multiparameter cytometry CRO services, combining the whole-blood preservation TokuKit with custom or off-the-shelf mass and spectral cytometry panels and analysis in the Teiko Dashboard. For clinical teams seeking flexibility without sacrificing quality, we provide a path to do more with every sample you collect.
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